JP2009022170A - Chloroplast pyruvic acid transporter protein and gene encoding the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chloroplast pyruvic acid transporter protein (TP1) closely involved in C4 photosynthesis circuit, and to provide a gene encoding the same. <P>SOLUTION: Provided are the protein as TP1 comprising a specific amino acid sequence, the protein comprising an amino acid sequence having ≥70% homology with the above amino acid sequence, a DNA comprising a specific base sequence as the gene comprising a domain encoding the TP1, and a DNA hybridizable, under stringent conditions, with a DNA comprising a base sequence complementary to the base sequence. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to proteins involved in the transport of pyruvate to chloroplasts, genes encoding the same, and uses thereof.

Some of the major grains and minor grains, such as corn, sorghum, sugarcane, millet and millet, have a metabolic circuit called the “C4 photosynthesis circuit”. Many have C4 photosynthetic circuits (8 of the 10 largest weeds in the world have). Pyruvic acid, a basic metabolite of plants, is deeply involved in the C4 photosynthesis circuit, and is important as a starting material for fatty acid synthesis, isoprenoid (IPP) synthesis, branched chain amino acid synthesis, etc. in chloroplasts. .

The C4 photosynthetic circuit is established by the division of labor between mesophyll cells and vascular sheath cells, and achieves a mechanism capable of concentrating carbon dioxide at a high concentration by metabolism between these two cells. Double photosynthetic activity (see FIG. 1). In mesophyll cells, the incorporated carbon dioxide reacts with phosphoenolpyruvate (PEP) to become oxaloacetate, which is further converted to malic acid or aspartic acid and transported to vascular sheath cells. C4 plants, mainly decarboxylase in bundle sheath cells NADP ⁺ - malic enzyme (NADP ⁺ -ME) or, NAD ⁺ - malic enzyme ^(NAD + -ME) or PEP carboxykinase by either a kinase (PCK) 3 For all these C4 circuits, pyruvic acid generated after decarboxylation in vascular sheath cells is known to move again into the chloroplasts of mesophyll cells and complete as a carbon circuit. Yes. However, although the existence of such a “pyruvate transporter” involved in the transport mechanism of pyruvate was suggested, the molecular entity was unknown.

In addition, it is known that the uptake of pyruvate into chloroplasts in mesophyll cells of plants is known to be promoted by the presence of sodium ions or hydrogen ions, and the pyruvate transporter involved is also different due to this difference. It is considered a thing. For example, corn (Zea mays
L.) and sorghum (Sorghum bicolor (L.) Moench), some NADP ⁺ -ME type C4 plants are hydrogen ion dependent, but NADP ⁺ -ME type C4 plants are also Echinochloa crus-galli (Echinochloa crus-galli ( L.) Beauv.) Is sodium ion dependent, and many other C4 plants such as NAD ⁺ -ME type C4 plant millet (Panicum miliaceum L.) are also sodium ion dependent (Non-patent Document 1). . In addition, the pyruvate transport ability of C3 plants is very low compared to C4 plants, extremely low values are measured in pea leaves, and transport activity is also measured in developing seeds of Brassica napa. However, no detailed biochemical analysis has been performed.
Naohiro Aoki, Jun-ichi Ohnishi and Ryuzo Kanai (1992): Two Different Mechanisms for Transport of Pyruvate into Mesophyll Chloroplasts of C4 Plants-a Comparative Study.Plant Cell Physiol. 33 (6), 805-809.

  An object of the present invention is to provide a chloroplast pyruvate transporter protein deeply involved in the C4 photosynthesis circuit and a gene encoding the same.

The present inventor has identified C3 species (Flaveria pringlei, etc.) and C4 species (F.
trinervia, etc.) is a technique for extracting genes with different gene expression levels.
Transcriptome analysis was performed using "Differential +/- method". As a result, a gene assumed to encode a chloroplast pyruvate transporter protein was found out of a plurality of genes that were highly expressed in C4 species, and the function of the protein was verified through verification of pyruvate transport activity. Thus, the present invention has been completed.

As a result of homology search by BLAST search, genes having sequences similar to the above FtTP1 gene are found in C3 plants Arabidopsis, tomato (Lycopersicon esculentum) and rice (Oryza).
sativa). Although the specific function of the protein encoded by these genes (Arabidopsis: At2g26900) has not been known so far, the experiment of the present invention revealed that the protein has a chloroplast pyruvate transport function. Surprisingly, it has also been found that in Arabidopsis, these genes are expressed only in the early stages of growth. In addition, even for C4 plants, it has been clarified that the above gene does not exist in plants having hydrogen ion-dependent pyruvate transport ability such as corn.

The present invention provides a chloroplast pyruvate transport protein corresponding to any of the following (I) to (V).
(I) a protein comprising the amino acid sequence represented by SEQ ID NO: 1;
(II) a protein comprising the amino acid sequence represented by SEQ ID NO: 2;
(III) a protein comprising the amino acid sequence represented by SEQ ID NO: 3;
(IV) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4;
(V) From an amino acid sequence modified by any one or more of amino acid deletion, substitution or addition within a range having 70% or more homology with any of the amino acid sequences of (I) to (IV) above A protein having the ability to transport chloroplast pyruvate.

In addition, the present invention provides an anti-peptide antibody obtained using a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 (part of the chloroplast pyruvate transport protein) as an antigen.
The present invention also provides a gene comprising a region encoding the above chloroplast pyruvate transport protein, for example, corresponding to any of the following (i) to (v).

(I) DNA comprising the base sequence represented by SEQ ID NO: 5;
(Ii) DNA consisting of the base sequence represented by SEQ ID NO: 6;
(Iii) DNA consisting of the base sequence represented by SEQ ID NO: 7;
(Iv) DNA consisting of the base sequence represented by SEQ ID NO: 8;
(V) It hybridizes under stringent conditions with DNA comprising a base sequence complementary to any of the base sequences (i) to (iv) above, and encodes a protein having a chloroplast pyruvate transport ability. DNA containing the region.

  Furthermore, the present invention provides a screening method for an activity inhibitor of the protein, which comprises the step of measuring the chloroplast pyruvate transport activity in the presence of the chloroplast pyruvate transport protein and the candidate substance. Such a method is particularly suitable for screening an activity inhibitor that can be used as an active ingredient of a herbicide.

  In this specification, the chloroplast pyruvate transport protein is collectively referred to as “TP1”, and in particular, TP1 of F. trinervia is “FtTP1”, TP1 of Arabidopsis is “AtTP1”, TP1 of rice is “OsTP1”, tomato TP1 is referred to as “LeTP1”, and the gene encoding these proteins is referred to as “TP1 gene”.

The TP1 protein and related genes, DNA, antibodies, and the like provided by the present invention are considered to greatly contribute to research and development related to chloroplast pyruvate transport protein.

  In particular, the TP1 gene of the present invention is expressed only in a C3 plant immediately after germination and a predetermined C4 plant, is not expressed in a grown C3 plant, and C4 plants such as corn do not have the gene. . Therefore, by screening for TP1 activity inhibitors, only strong weeds are killed without adversely affecting the growth of cultivated crops, and there is no risk of harm to humans or animals without chloroplasts. It opens the way for the development of highly useful herbicides.

The chloroplast pyruvate transporter protein and its DNA represented by SEQ ID NOs: 5, 6, 7 and 8 are C4 species of the genus Flavelia (F. trinervia), Arabidopsis thaliana (A. tahliana), rice (O sativa) and tomato (L. esculentum), FtTP1 consisting of the amino acid sequence represented by SEQ ID NO: 1, AtTP1 consisting of the amino acid sequence represented by SEQ ID NO: 2, and represented by SEQ ID NO: 3, respectively. A gene encoding a region encoding OsTP1 consisting of the amino acid sequence and LeTP1 consisting of the amino acid sequence represented by SEQ ID NO: 4.

  The protein encoded by the TP1 gene is responsible for sodium-dependent chloroplast pyruvate transport function. As shown in the examples described later, this indicates that the pyruvate transport activity to sodium ion-dependent chloroplasts of wild-type Arabidopsis thaliana (cotyledons and first and second true leaves) is TP1 gene Disappearance in the disrupted strain, that the transport activity of tobacco overexpressing the TP1 gene is increased compared to the wild type, and that the TP1 gene is actually highly expressed in the daytime leaves of C4 plants, It can be strongly estimated from the fact that it becomes localized in the chloroplast of mesophyll cells. It should be noted that the chloroplast pyruvate transport protein of the present invention is strongly predicted to be a membrane protein of the chloroplast inner membrane.

  Such a TP1 gene is expressed in a large amount in the leaves of C4 plants, while it is hardly expressed in the fully expanded leaves of C3 plants. Therefore, C3 and C4 plants belonging to the same genus as shown in the Examples below. Can be obtained by differential screening.

  The differential screening, accompanying mRNA extraction, cDNA production and other operations can be performed according to known methods, and are not particularly limited. One aspect of this is as described in the examples below.

  In addition, DNA probes are synthesized based on the FtTP1 gene obtained as described above, and those TP1 genes are obtained by screening cDNA libraries or genomic libraries of other plants using this. can do.

  The DNA containing the TP1 gene of the present invention is not limited to those derived from nature, but also includes chemically synthesized DNA, PCR products using the TP1 gene, etc., and the DNA is a single strand DNA of the sense strand alone. Even if it exists, it may exist as double-stranded DNA consisting of a sense strand and an antisense strand. In addition to the base sequence encoding the TP1 protein (TP1 gene), it may contain a region for transcription control (enhancer, promoter, etc.) and an untranslated region.

  FIG. 2 is a diagram comparing the amino acid sequences of FtTP1, AtTP1, and OsTP1. The amino acid sequences of the proteins of these three types and LeTP1 (not shown in the figure) have a homology of about 70% as a whole, but the portion corresponding to about 100th or more from the N-terminal side of FtTP1 is particularly high. This part may be important for the activity of the protein. Therefore, it is a protein having an amino acid sequence having 70% or more, preferably 90% or more, more preferably 95% or more homology with the above four types of amino acid sequences, and more preferably only one or several amino acid sequences. In particular, those having the same homology among the above four amino acid sequences are considered to have the function of transporting pyruvic acid to the chloroplast.

  In addition to DNA consisting of the base sequences represented by SEQ ID NOs: 5 to 8, DNA that hybridizes under stringent conditions with DNA consisting of base sequences complementary to any of these base sequences is also the TP1 gene. Is a DNA that can encode Here, the “stringent condition” means a condition in which a so-called specific hybrid is formed and a non-specific one is not formed. The sodium concentration is 150 to 900 mM, preferably 600 to 900 mM, and the temperature is 60 to The conditions are 68 ° C, preferably 65 ° C.

Screening method By using TP1 provided by the present invention, it is possible to screen for substances that affect its function.

  For example, in the presence of TP1 and a candidate substance, the pyruvate uptake rate of chloroplasts is measured, and the effect of TP1 is evaluated by evaluating the effect compared to the rate of pyruvate uptake of normal chloroplasts Inhibitors can be screened.

  Moreover, the substance with strong TP1 activity inhibitory effect obtained by such screening is useful as an active ingredient of a herbicide. For example, an acetolactate synthase inhibitor involved in the synthesis of branched-chain amino acids is already used as a herbicide in a series of metabolisms starting from pyruvic acid in the chloroplast. However, pyruvate transport is located at a higher metabolic level than this, and it is considered that it affects other metabolisms starting from pyruvate, so that it can be expected to be more effective than an acetolactate synthase inhibitor.

  As a step related to the above screening method, analysis of protein secondary structure (domain, motif), higher order structure, and other structural features using a computer or the like based on the amino acid sequence information of TP1. Thus, it is also possible to narrow down compounds that can become TP1 activity inhibitors.

Anti-peptide antibody In one aspect, the present invention provides an antibody that specifically binds to TP1. Such an antibody can be obtained by a known method for producing a polyclonal antibody or a monoclonal antibody using TP1 as an antigen. For example, an antibody obtained using a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 (the amino acid sequence of the 1st to 12th amino acids on the C-terminal side of FtTP1) as an antigen is excellent in specific binding properties with TP1, and various analyzes It can be used for.

For cloning differential screening of the FtTP1 gene, the F. trinervia whole leaf cDNA library was used as the cDNA library. As probes, two kinds of probes, ie, a probe derived from F. pringlei whole leaf RNA and a probe derived from F. trinervia whole leaf RNA were used. A method for producing the probe will be described later. The specific screening method is as follows. This library is incorporated into λgt10 phage, infected with Escherichia coli (NM514), and plaques are grown to about 1,000 clones per 9 cm × 12 cm plate, so that a total of 9,000 clones are prepared. A plate was prepared. Phages were transferred from these plates to nylon membranes to produce two sets of identical membranes. Amersham Pharmacia HybondN + was used as the membrane, and the membrane preparation and hybridization procedures followed the HybondN + membrane protocol. The method is as follows. First, put HybondN + membrane on the agar medium where the phages are grown, adhere for 30 seconds (1 minute for the second sheet), peel off the membrane, and denature the solution with the plaque side up (1.5 M NaCl, 0.5 M NaOH) Placed on filter paper soaked for 7 minutes. Next, place it on a filter paper soaked in neutralization solution (1.5 M NaCl, 0.5 M Tris-HCl (pH 7.2), 1 mM EDTA) for 3 minutes, and add 2 × SSPE (0.36 M NaCl, 20 mM sodium phosphate). (pH 7.7), 2 mM EDTA). Thereafter, the phage DNA was fixed on the membrane by placing on a filter paper soaked in 0.4 M NaOH for 20 minutes, and washed with 2 × SSPE. Add the membrane and hybridization solution (5 × SSPE, 5 × Denhardt, 0.5% SDS, 20 mg / ml salmon sperm DNA) to Hybridback (Cosmo Bio) and prehybridize at 60 ° C for 1 hour or longer. went. Furthermore, a labeled single-stranded cDNA probe synthesized using poly (A) + RNA as a template was added, and hybridization was performed while gently shaking at 65 ° C. for 2 days.

The labeled single-stranded cDNA used as a probe was poly (A) + RNA (described later) 1.5 μg as a template, Amersham Pharmacia's [α- ³² P] dCTP, and BIO-RAD's M-MuLV Reverse Transcriptase. Was synthesized. After reverse transcription reaction, unreacted [α- ³² P] dCTP was removed through Sephadex-G50 spun column, purified by phenol / chloroform extraction and ethanol precipitation, and dissolved in 200 μl of sterilized water. The specific radioactivity of the synthesized probe was 4.2 × 10 ⁸ cpm / μg for the probe prepared from F. trinervia mRNA and 2.4 × 10 ⁸ cpm / μg for the probe prepared from F. pringlei mRNA. These probes were used at a concentration of approximately 10 ⁶ cpm / ml.

The membrane was washed once with 400 ml of washing solution I (2 x SSPE, 0.1% SDS) at 60 ° C for 15 minutes, and with washing solution II (0.2 x SSPE, 0.1% SDS) at 60 ° C for 15 minutes. I went with shaking once. After washing, the membrane was brought into close contact with an imaging plate of Fuji Film for 16 hours, and radioactivity was analyzed by imaging using a bioimaging analyzer BAS2000. After identifying strong signals with the F. trinervia mRNA probe, the corresponding phage plaques were recovered from the plates. After this, the membrane used for differential screening was deprobed and phosphoenolpyruvate carboxylase (PpcA), pyruvate dikinase (PPDK) and NADP-malic enzyme (NADP-ME), which are representative of C4 type enzymes The partial sequence was amplified by Polymerase Chain Reaction (PCR), and hybridization was carried out under the same conditions as described above with a labeled probe prepared using the obtained DNA fragment as a template. For PCR, TaKaRa Ex taq was used. The C4 type enzyme signal read by the imaging plate was compared with the C4 high expression signal obtained by the differential screening, and it was confirmed that it was isolated as a high expression signal. Thereafter, the signal of the C4 type enzyme was excluded from the analysis.

A series of procedures isolated 126 clones that exhibited specific signals in the C4 species, and further classified them into 26 groups based on their homology. The nucleotide sequence was determined using an ABI PRISM ™ 310 automatic sequence analyzer from PE Applied Biosystem.
This is a method for determining a base sequence based on the mobility of a DNA fragment labeled with a fluorescent dye.
ABI PRIMTM Dye Terminator Cycle Sequencing for sample preparation for base sequencing
Ready Reaction Kit was used. First, a reaction mixture containing 4 types of fluorescently labeled dideoxynucleotides and Taq polymerase (Terminator Ready Reaction Mix 4 μl, template 0.2
(μg, primer 1.6 pmol, sterile distilled water 2.4 μl) 10 μl was overlaid with 20 μl of mineral oil, and PCR was performed. For PCR, TaKaRa PCR Thermal Cycler was used, and a cycle of 96 ° C. for 30 seconds, 50 ° C. for 15 seconds, and 60 ° C. for 4 minutes was repeated 25 times. The sample after the reaction is Ethanol Precipitation
Purification was performed according to Protocol 2, and automatic analysis was performed using ABI PRISMTM 310. GENETYX Mac manufactured by SDC Software Development Co., Ltd. was used for DNA base sequence analysis.

Subsequently, a homology search using BLAST (Basic Local Alignment Search Tool) was performed. As a result, one of the clones described above was associated with a protein related to “bile acid sodium symporter family protein” that could be assumed to be a pyruvate transporter. A gene encoding (named FtTP1) was discovered. It was also found that genes with similar sequences exist in Arabidopsis and rice. These three types of TP1 are shown in FIG.

  It was confirmed that the above-described differential screening was functioning together with the marker gene PpcA (one of the above clones, a gene already known for C4 plants) and other clones.

The clones that had undergone gene sequence analysis after grouping were subjected to Northern hybridization to confirm the accuracy of differential screening. C4 type, C3 type
At the same time, the expression in the intermediate species F. ramosissima was also investigated. As the DNA fragment used for the probe, the entire inserted cDNA region of each clone isolated from the cDNA library was mainly used. 10 μg of each RNA was subjected to 1% formaldehyde gel electrophoresis. At the same time, an RNA ladder marker (0.24-9.5 kb) (GIBCO BRL) was run, and after electrophoresis, it was used to determine the size of mRNA by staining with ethidium bromide. Membrane is Hybond N + as well as screening
A membrane was used. Transfer RNA from gel to membrane and hybridization procedure according to the protocol, transfer for 12 hours with 20 x SSPE, and place membrane on Whatman 3MM filter paper soaked with 0.05N NaOH for 5 minutes. RNA was fixed. After the alkali treatment, it was neutralized by shaking in 2 × SSPE for 10 to 60 seconds.

Prepare a hybridization solution (5 × SSPE, 5 × Denhardt solution, 0.5% SDS) to a 1 ml / 20 cm ² membrane, and heat-denatured salmon sperm DNA to a final concentration of 20 mg / ml. In addition, the solution and membrane were immersed in the hybrid bag and left at 60 ° C. for 1 hour or longer. Meanwhile, a probe was prepared from 12.5 ng of the template DNA fragment using Amersham [α- ³² P] dCTP and Megaprime DNA labeling system. This probe was added to the hybridization solution, and hybridization (overnight at 60 ° C.) was performed with gentle shaking. After hybridization, washing was performed under the same conditions as described above. The radioactivity bound to the membrane after the treatment was measured with an image analyzer and analyzed as a relative value. The membrane once used was immersed in a 0.5% (w / v) SDS solution at 100 ° C. and left to room temperature to remove the probe and reuse it.

State of expression of FtTP1 gene In order to confirm the expression of the FtTP1 gene in plants, the following experiment was conducted.
-RNA gel blotting In order to investigate the expression of FtTP1, RNA prepared from roots or stems was used together with leaves in light and dark periods. The analysis procedure and conditions are as described above.

The results are as shown in FIG. From the above experiments, it was found that FtTP1 is expressed in a large amount in the daytime leaves of C4 species (F. trienervia).
・ In situ hybridization
In situ hybridization was performed to investigate more specific tissue-specific expression. First, the preparation of leaf samples for observation of expressed tissues will be described. The leaf tissue was cut to 5 mm x 5 mm with dissecting scissors and immediately infiltrated with 15 ml of formaldehyde fixative in a 20 ml glass vial. The fixative was replaced with 0.05 M Na-P buffer and gently washed for 30 minutes for washing. After performing this washing twice, the ethanol series was dehydrated with gentle shaking at room temperature and replaced with 100% t-butanol. Thereafter, paraffin (Paraplast, OXFORD) liquefied under a temperature condition of 60 ° C. was overlaid, and t-butanol was evaporated to obtain a paraffin-fixed sample.

Using a microtome, a continuous section was cut out to a thickness of 10 μm from a paraffin-embedded block to produce a paraffin ribbon.
The slide glass used for hybridization was selected under a light microscope as a set of two pieces with continuous sections attached, placed on a hot plate at 60 ° C to dissolve paraffin, and immersed in 100% xylene twice for 10 minutes. Paraffin was dissolved. Next, replace xylene with ethanol (50% xylene / ethanol for 5 minutes, 100% ethanol twice for 5 minutes), dry under reduced pressure for 1 hour, and then ethanol descending series (100, 90, 70, 50, 30% ethanol 2 Min) and sterilized water for 5 minutes twice to replace ethanol with water. Next, proteinase K treatment was performed to increase the tissue permeability of the probe. Treatment solution (100 mM Tris-HCl, 50 mM
EDTA-2Na, 5 mg / ml Proteinase K, pH 7.5) was first self-digested in a 37 ° C. water bath (YAMATO SCIENTIFIC) for 20 minutes, and a slide glass was immersed in it and treated for 30 minutes. After treatment, wash 3 times with sterilized water at room temperature for 5 minutes, then re-fix with refix solution (4% PFA, 10 mM Na-P buffer) for 10 minutes, and then wash with sterilized water 3 times for 5 minutes. did. In order to reduce nonspecific adsorption of the probe, the tissue piece was acetylated with acetylated solution (0.1 M triethanolamine, 0.25% acetic anhydride) for 10 minutes, and 2 × SSPE (20 mM NaH ₂ PO ₄ , 0.3 After washing twice with M NaCl, 2 mM EDTA-2Na, pH 7.4) for 5 minutes each, ethanol ascending series (30, 50, 70, 90% ethanol for 2 minutes each), 100% ethanol for 5 minutes with 2 treatments It dehydrated and dried under reduced pressure for 1 hour.

  200 μl of hybridization solution per slide glass (50% formamide, 300 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA-2Na, 1 × Denhardt solution, 60 mM DTT, 1 mg / ml yeast tRNA, 500 μg / ml poly (A), 10% dextran sulfate, pH 7.5) was prepared, heated at 80 ° C. for 5 minutes, and then 6 μl of a probe denatured by rapid cooling on ice was added. Slide glasses were arranged in a wet box containing 50% formamide, 2 × SSC (0.3 M NaCl, 30 mM Citrate-3Na) as a wet solution, and about 180 μl of hybridization solution per slide glass was placed on the section. A pair of slide glasses was loaded with a sense probe on one side and an antisense probe on the other. Thereafter, a cover glass was applied so that the liquid spread over the entire slide glass. The wet box was sealed and hybridization was performed at 50 ° C. overnight.

Thereafter, the slide glass was set on a staining rack and washed with the solution placed in the staining basket. First, wash with 4 x SSC in a 50 ° C water bath for 15 minutes,
The sample was carefully removed so as not to peel off. Thereafter, washing with 4 × SSC was repeated 3 times for 5 minutes each at 50 ° C. Next, RNase treatment was performed to degrade single-stranded RNA. RNase A was added to RNase Buffer (16.2 mM Tris-HCl, 5 mM EDTA-2Na, 500 mM NaCl, pH 7.5) to a concentration of 20 μg / ml, and incubated in a 37 ° C. water bath for 30 minutes. Then, wash with RNase buffer three times for 15 minutes each at 37 ° C, then use 0.5 × SSC twice at 50 ° C for 20 minutes each time with Buffer1 (100 mM Tris-HCl, 150 mM NaCl, pH 7.5). The plate was washed twice for 5 minutes at room temperature with gentle stirring with a stirrer.

Slide glass removed from Buffer1 is placed in a damp chamber with distilled water as a wetting solution, and blocking solution (50% Normal rabbit serum, 50 mM Tris-HCl, 75 mM NaCl, 0.5% Tween 20, pH
7.5) was applied to each plate at about 200 μl and incubated at room temperature for 30 minutes for blocking. Tilt the glass slide and gently wipe the blocking solution.500 μl of Anti-DIG Alkaline Phosphatase (AP) solution (0.1% BSA, 100 mM Tris-HCl, 150 mM NaCl, 0.375 U Anti-DIG-AP (Roche Diagnostics), pH 7.5) was added, and the antibody reaction was performed at room temperature for 2 hours. After completion of the reaction, wash the slide glass one by one with Buffer 1 using a washing bottle, and then add Buffer 1 into the staining basket three times for 10 minutes while stirring with a stirrer, and then Buffer 3 (100 mM Tris-HCl, The plate was washed with 100 mM NaCl, 50 mM MgCl ₂ , pH 9.5) for 5 minutes.

  After washing, place slide glass in a new damp chamber moistened with sterilized water, and add 200 μl of color reaction solution (NBT / BCIP stock solution (DIG nucleic acid detection kit, Boehringer Mannheim) to 10 ml of Buffer3) ) Was covered with about 500 μl per slide glass, and the color was developed by shading. The state of color development was observed with a microscope. When color development was observed, a pair of slide glasses labeled with a sense probe and an antisense probe was simultaneously washed with TE and then with sterile water for 5 minutes each to stop the color reaction. After that, it was dehydrated in an ethanol ascending series, and ethanol was replaced with xylene (100% ethanol twice for 5 minutes each and xylene for 5 minutes twice), and the cover glass was mounted. The encapsulant was ENTELLAN neu (MERCK), dried in a well-ventilated place overnight and observed under an optical microscope.

The results are as shown in FIG. From the above experiment, it was found that FtTP1 is expressed in mesophyll cells of C4 species (F. trienervia).
・ Fluorescent labeling with GFP
Preparation of FtNBAT-: GFP construct: The plasmid for gene transfer was inserted with cauliflower mosaic virus 35S promoter (CaMV35S), synthetic green fluorescent protein (sGFP: S65T) gene, and noparine syntase (nos) 3 terminator at the pUC18 multicloning site. I used something. FtTP1 coding region was used as the transgene, a site mutation was introduced into the Nco I site in FtTP1 by PCR, and Nco I sites were added to both ends. However, this time, the primer was designed so that the stop codon of FtTP1 was converted and translation was not terminated there, and the reading frame with sGFP was not shifted.

A: CTTCccatggCGTCTATTTCAAGTATTG (ccatgg: Nco I site)
Cf: GTTTAAGGAATCCgTGGACTGTAGGTGTAG (mutation: A → g)
Cr: CTACACCTACAGTCCAcGGATTCCTTAAAC (mutation: T → c)
F: GCccatggACTCCTTGAAATCATCTTTGTC (ccatgg: Nco I site)
The gene was introduced into the plant leaves using Bio-Rad Biolistic (registered trademark) PDS-1000 / He Particle Delivery System (Bio-Rad) according to the protocol. Tungsten particles were washed with 70% EtOH and rinsed 3 times or more with sterile water. Finally, it was suspended with sterilized 50% glycerol to 60 mg / ml. To 1.5 mg of the treated tungsten particles, 5 μg plasmid, 0.625 mM CaCl2, 10 mM spermidine (3 shots) was added with vigorous stirring by vortexing, and the mixture was stirred as it was for 3 minutes. The supernatant was removed by light centrifugation, rinsed sequentially with 70% and 100% ethanol, and suspended in 30 μl of 100% ethanol. A 10 μl dose was dropped onto a macrocarrier, allowed to air dry, and then poured into a plant body. The rupture disk was 1350 PSI, the degree of vacuum in the chamber was 28 inch Hg, and the distance between the stopping plate and the sample was 4 cm. The tissue into which the gene was introduced was tobacco leaves grown in a greenhouse for about 4 weeks. The leaves were cut into 3 cm squares, introduced into MS plates (1 × Murashige-Skoog, 1% (w / v) agar), and induced for expression. After the gene introduction, the cells were cultured overnight at 25 ° C. in the dark and observed. GFP fluorescence was observed after culturing using a fluorescence microscope (Nikon ECLIPSE E600) at an excitation wavelength of 490 nm and an observation fluorescence wavelength of 520 nm or 520-560 nm.

  The results are as shown in FIG. From the above experiment, it was found that FtTP1 is also transported to chloroplasts in tobacco leaves, indicating that this protein is involved in substance transport to chloroplasts.

Detection of FtTP1 using anti-peptide antibodies For the purpose of investigating the generality of TP1 in C4 plant species, anti-peptide antibodies using a part of TP1 as an antigen were prepared. In addition to the aforementioned F. trienervia, Cleome gynandra , Corn (Zea mays), millet (Panicum miliaceum) and other C4 plants, as well as the aforementioned F. pringlei, Cleome spinosa, rice (Oryza s)
Ativa) and other C3 plants were examined for the presence or absence of proteins crossed by this antibody.

  The antigen region was defined as a C-terminal charged region that was fluorescently labeled in FIG. Anti-peptide antibodies were made by a versatile technique for immunizing rabbits by requesting the Peptide Institute.

  The results are as shown in FIG. In the above experiment, C4 showed high cross-reactivity with C. elegans and millet having sodium ion-dependent pyruvate transport ability, but no cross-reactivity appeared in corn with hydrogen ion-dependent pyruvate transport ability. This suggests that TP1 is an entity exhibiting sodium ion-dependent pyruvate transport ability. In addition, the crossing property was shown regardless of monocotyledon (monocot) or dicotyledonous (dicot).

Subsequently, using the above anti-peptide antibody, an immunohistochemical analysis was conducted to investigate the localization of a protein having a cross-reactivity with this antibody.
The technique was based on performing antigen-antibody reaction on paraffin sections prepared according to the in situ hybridization section. The presence of the antibody was determined by gold colloid silver sensitization using a goat anti-rabbit antibody as a secondary antibody.

The results are as shown in FIG. It has been shown that a protein (ie, TP1) that exhibits cross-reactivity with the anti-peptide antibody is localized in the chloroplast of mesophyll cells.
Expression of TP1 gene in Arabidopsis thaliana In order to investigate the expression of TP1 gene in Arabidopsis thaliana, which is a C3 plant, the following experiment was conducted.

For transformation of Arabidopsis thaliana, the pBI101-GUS-nosT vector was used, and 2 kb upstream from the translation initiation Met of AtTP1 (At2g26900) was introduced upstream of the β-glucuronidase (GUS) gene.
First, we obtained the base sequence information of the upstream region of AtTP1 from the genome information of Arabidopsis thaliana. Therefore, using the Arabidopsis genomic DNA as a template, about 2 kb upstream was amplified by PCR immediately before the start codon of AtTP1. The primers were designed to introduce a Hind III site on the 5 ′ end side and an Xba I site on the 3 ′ end side. For the PCR reaction, 3 ng of template and Extaq polymerase were used, and the reaction conditions were denaturation at 94 ° C / 30 seconds, annealing at 55 ° C / 2 minutes, and extension at 72 ° C / 1 minute for 35 cycles. The primer sequences used are listed below.

M: 5'- CGaagcttGGCCTGTTTTGATCAAAATCA -3 '(aagctt: Hind III site)
N: 5'- CGtctagaGTTTTGATCAAAAGGGTTTTAG -3 '(tctaga: Xba I site)
The product amplified by PCR was treated with restriction enzyme with Hind III / Xba I and then separated by agarose gel electrophoresis. After complete separation, the gel was cut out from the gel, purified using MagExtractor (TOYOBO), and a subcloning product was obtained according to a conventional method. Hind this subcloning product
III / Xba I digested and recovered from agarose gel by the above method. This was introduced into the upstream of the GUS gene of the pBI101 vector recovered by digestion with Hind III / Xba I in the same manner.

  A transformed Agrobacterium was prepared using GV3101 :: pMP90 as a strain of Agrobacterium tumefaciens, and transformed into Arabidopsis thaliana by the commonly used vacuum infiltration method. Eventually, a strain in which the transgene was one copy and was homozygous was obtained. Whole-mount GUS staining was performed for analysis of GUS activity. GUS staining buffer (0.3% Triton X-100, 1.9 mM 5-bromo-4-chloro-3-indoyl-β-D-glucronide, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, 100 mM phosphate buffer, pH 7.0) was infiltrated. It put in the desiccator and pressure-reduced for 1 hour at 400 mmHg. Thereafter, the reduced pressure was released slowly, and the mixture was incubated at 37 ° C. for 24 hours. After that, remove the GUS staining buffer and stop the reaction by washing the sample once with 75% ethanol, then infiltrate the sample in fresh 75% ethanol and incubate at 4 ° C for 24 hours to remove chlorophyll. Elute and decolorize.

  The results are as shown in FIGS. The first and second true leaves of Arabidopsis thaliana are colored blue, indicating that the AtTP1 gene is expressed. On the other hand, the third true leaf (in the frame of FIG. 9) and the root are not colored blue, suggesting that the AtTP1 gene is not expressed in the growth stage after the third true leaf.

Subsequently, whether the TP1 protein was present at the time of limited expression as described above was examined using the aforementioned anti-peptide antibody.
Here, regarding the Arabidopsis AtTP1 gene, it has been found by the above-mentioned BLAST search that there are two independent gene-disrupted strains by insertion of T-DNA (SALK_098692 and SALK_101808). AtTP1 mRNA and protein were detected from these mutants and wild type.

  Proteins or RNAs were extracted from individuals who started to develop the first second true leaves, and each was detected using a TP1 antibody or a radiolabeled AtTP1 gene fragment.

  The results are as shown in FIG. In the germinating wild type, both AtTP1 mRNA and protein were detected, but in the two disrupted strains, neither AtTP1 mRNA nor protein was detected, confirming that the AtTP1 gene was destroyed. It was.

Measurement of pyruvate transport activity It was proved by the following two experiments that TP1 is a protein responsible for the function of transporting pyruvate to chloroplasts.

  Isolated chloroplasts were prepared from Arabidopsis thaliana, wild strains, and gene-disrupted strains at the above growth stage, and the radiolabeled pyruvate transport activity was investigated by the silicon double layer method. By preparing a solution containing or not containing 50 mM sodium in the intermediate layer of the double layer, the requirement for sodium ions can be examined. The procedure is as described in the non-patent literature shown above.

  The results are as shown in FIG. The wild-type strain showed sodium ion-dependent pyruvate transport activity, but the disrupted strain disappeared, indicating that AtTP1 is a chloroplast pyruvate transport protein.

Next, a tobacco transformant (FtT) into which FtTP1 fused with the GFP gene was introduced.
P1 was overexpressed), and the pyruvate transport activity was compared with tobacco at the same time (about 7 days of age). Preparation of chloroplasts and measurement of pyruvate uptake rate were carried out in the same manner as in the experiment for Arabidopsis thaliana.

  The results are as shown in FIG. In the transformant (G2), it is observed that pyruvate transport activity increases in a sodium ion-dependent manner, and it is considered that overexpression of FtTP1 is reflected.

The schematic diagram of a C3 and C4 photosynthesis circuit. Comparison of amino acid sequences of FtTP1, AtTP1 and OsTP1. RNA blotting of FtTP1. It can be seen that the amount of expression in daytime leaves of C4 species is large. In Situ hybridization of FtTP1. It can be seen that it is expressed in mesophyll cells. Expression of TP1 :: GFP gene in tobacco leaves. The chloroplast is localized, indicating that TP1 contributes to mass transport into the chloroplast. Crossability test using anti-peptide antibody. It shows high cross-reactivity with the C4 species exhibiting sodium ion-dependent pyruvate transport activity. Detection of TP1 by immunohistochemistry. Localized in the chloroplast of mesophyll cells. Expression of the AtTP1 gene during the emergence of Arabidopsis thaliana introduced with the GUS gene. Expression of AtTP1 gene in Arabidopsis thaliana introduced with GUS gene. The first and second true leaves are colored blue, but the third true leaf (within the frame) and the root are not colored. AtTP1 mRNA and protein detection experiments on wild and disrupted Arabidopsis strains. Only wild type was detected. Measurement of pyruvate uptake rate into chloroplasts of Arabidopsis thaliana. In the wild type, pyruvate transport activity is increased in a sodium ion-dependent manner, but this is not seen in the disrupted strain. Measurement of pyruvate uptake rate into tobacco chloroplasts. In plants overexpressing FtTP1, pyruvate transport activity is increased.

Claims (6)

A chloroplast pyruvate transport protein corresponding to any of the following (I) to (V).
(I) A protein comprising the amino acid sequence represented by SEQ ID NO: 1 (II) A protein comprising the amino acid sequence represented by SEQ ID NO: 2 (III) A protein comprising the amino acid sequence represented by SEQ ID NO: 3 (IV) SEQ ID NO: (V) Any of amino acid deletion, substitution or addition within the range having 70% or more homology with any one of the amino acid sequences (I) to (IV) above A protein comprising an amino acid sequence modified by one or more species and having a chloroplast pyruvate transport ability   A gene encoding the protein according to claim 1. A gene comprising a region encoding the protein according to claim 1, which corresponds to any of the following (i) to (v).
(I) DNA comprising the base sequence represented by SEQ ID NO: 5
(Ii) DNA comprising the base sequence represented by SEQ ID NO: 6
(Iii) DNA comprising the base sequence represented by SEQ ID NO: 7
(Iv) DNA comprising the base sequence represented by SEQ ID NO: 8
(V) Hybridizes under stringent conditions with DNA comprising a base sequence complementary to any of the base sequences (i) to (iv) above, and encodes a protein having a chloroplast pyruvate transport ability DNA containing region   The screening method of the protein activity inhibitor of Claim 1 including the process of measuring the pyruvate transport activity of a chloroplast in presence of the protein of Claim 1, and a candidate substance.   The screening method according to claim 4, wherein the activity-inhibiting substance can be used as an active ingredient of a herbicide.   An anti-peptide antibody obtained using a peptide consisting of the amino acid sequence represented by SEQ ID NO: 9 as an antigen.